There is presently unclear need for the development of new markers for the diagnosis of diseases associated with elevated levels of proteolytic cleavage of proteins in the body, particularly those involved in the early stages of tumorigenesis. Tumor development is accompanied by a high level of proteolytic activity (Al-Majid S., Waters H. The biological mechanisms of cancer-related skeletal muscle wasting: the role of progressive resistance exercise/Biol. Res. Nurs. 2008. Vol. 10, No 1. pp. 7-20). Indeed, the level of proteolytic activity is currently considered one of the factors of carcinogenesis (Bashir T., Pagano M. Aberrant ubiquitin-mediated proteolysis of cell cycle regulatory proteins and oncogenesis/Adv. Cancer. Res. 2003. Vol. 88. pp. 101-144). Several types of proteases are involved in the process of carcinogenesis by enhancing the proliferation, invasion, and metastasis of tumor cells (Chilingirov AD proteolysis inhibitor effect on some bacterial pathogens, and for inflammatory processes/Pat. Fiziol. And experimental. Therapy. 1997. No 3. C. 37-39; Søreide K. Proteinase-activated receptor 2 (PAR-2) in gastrointestinal and pancreatic pathophysiology, inflammation and neoplasia/Scand. J. Gastroenterol. 2008. Vol. 43, No 8. pp. 902-909). Of these, serine proteases are reported to most significantly contribute to the process of carcinogenesis (Zorio E., Gilabert-Estellés J., Españia F., Ramón LA, Cosín R., Estellés A. Fibrinolysis: the key to new pathogenetic mechanisms/Curr. Med. Chem. 2008. Vol. 15, No 9, pp. 923-929).
Serine proteases usually cleave peptide bonds between positively charged amino acids lysine and arginine, as well as the esters and amides of these amino acids (Fersht E. The structure and mechanism of action of enzymes. Ed. Kurganova BI Moscow, “Mir” 1980. 432 pp . . . ). To date, some authors have shown that the products of proteolytic activity can serve as a universal marker, the detection of which is associated with various autoimmune and oncogenic processes. For example, the high content of the products of proteolytic cleavage of immunoglobulins can be used as a marker of autoimmune disease or cancer (Robert Jordan et al, U.S. Ser. No. 08/501,907). In that work, the authors proposed a method for the detection of proteolytic cleavage of immunoglobulins using polyclonal and monoclonal antibodies. Other authors presented data on the specific proteolysis of immunoglobulins by plasmin (Peter S. Harpel et al The J. of biological chemistry Vol. 264, No. 1, Issue of January 5, pp. 616-624 (1989)). Following cleavage, the immunoglobulin molecule was shown to specifically interact with plasminogen due to the presence of a C-terminal lysine.
Plasmin, a trypsin-like serine protease, is usually generated by the activation of plasminogen by streptokinase, urokinase or tissue plasminogen activator (tPA). It is well known that plasminogen exhibits fibrinolytic activity and can block clot formation by binding to the C-terminal lysine residues of fibrin and the proteolysis of fibrin fibers. Both plasminogen and plasmin bind to fibrin through kringle regions, each of which is a triple loop region formed by disulfide bonds. Kringles K1, K2, K3, K4, and K5 have a strong affinity for lysine.                a. The participation of the C-terminal lysine in protein binding to plasminogen has been demonstrated by Marco Candela et al., (Binding of Human Plasminogen to Bifidobacterium, Journal of bacteriology, August 2007, p. 5929-5936). In this study, proteins which bind to plasminogen were treated with carboxypeptidase B, which specifically cleaves only C-terminal lysine and arginine. After this treatment, the proteins lost their ability to bind to plasminogen, indicating that C-terminal lysine participation is essential for the binding to plasminogen and its fragments.        
The plasminogen/plasmin system not only takes an active part in the process of fibrinolysis, but has also been shown to be closely associated with angiogenesis and carcinogenesis. Interestingly, some products of plasminogen degradation may be more active than the intact plasminogen molecule in the processes of angiogenesis and carcinogenesis. (Y G Klys, et all, Proteolytic plasminogen derivatives in the development of malignancies, Oncology, V 12, No. 1, 2010). The following variants of fragments of plasminogen in the plasma have been described: K1-3; K2-3; K1-4; K1-4, 5; and K1-5 (Perri S, Martineau D, Francois M, et al. Plasminogen kringle 5 blocks tumor progression by antiangiogenic and proinflammatory pathways. Mol Cancer Ther 2007; 6: pp. 441-449). It has previously been shown that all kringle domains are actively involved in angiogenesis and carcinogenesis. The activity of the first four kringles (K1-4) is the best-studied—they play a role in angiogenesis. For example, this sequence of kringle domains is found in angiostatin (Francis J. Castellino, Victoria A. Ploplis, Structure and function of the plasminogen/plasmin system, ThrombHaemost 2005; 93: pp. 647-54; C. Boccaccio and Paolo M. Comoglio Cancer Res 2005; 65 (19): pp. 8579-82; Rijken D C, Lijnen H R. New insights into the molecular mechanisms of the fibrinolytic system. J Thromb. Haemost 2009; 7: pp. 4-13). The activation of plasminogen and some of the other serine proteases leads to an increase in the amount of C-terminal lysine containing protein proteolysis products during carcinogenesis. Since the intact plasminogen molecule as well as its fragments has lysine binding sites, they can bind the degradation products with a C-terminal lysine generated by serine proteases and be used as detectors of the process of carcinogenesis and other pathological processes. This detector has universal properties compared with other proposed methods of detecting degradation products, which require using monoclonal antibodies specific for each product of proteolysis.
The detection of the products of proteolysis in plasma in both human and animal samples can be performed using enzyme-linked immunosorbent assay (ELISA), where the plasminogen molecule or its fragments are used as a detector. The ELISA was first developed in 1971 and currently, an extensive range of types and modifications of ELISA are used. The basic principles of ELISA, regardless of modifications, are as follows:                1. At the first stage of the reaction, antigens or antibodies are adsorbed onto a solid phase. The reagents or compounds not bound to the solid phase are easily removed by washing.        2. Test samples and controls are incubated in the coated wells—thus, immune complexes can be formed on the surface of the solid phase. Unbound components are removed by washing.        3. Antibody-enzyme or antigen-enzyme conjugates, which bind a complementary site on the antigen (or antibody) on the solid phase are then added. Their binding is detected via a colorimetric reaction after the adding of the substrate for the conjugated enzyme. This reaction can be stopped and optical density can be measured.        
The levels of the immunoglobulin and other proteins after proteolysis are determined using an indirect ELISA. The wells are coated by antibodies to the desired protein (antigen) and incubated with the serum (plasma) samples or other biological material from the patient (cerebrospinal fluid, saliva, etc.). Specific antigens bound to antibodies at the solid phase are detected using a second antibody-enzyme conjugate to another epitope of the antigen. Depending on the purpose of the assay, different antigens are used, either with antibodies universal for all isotypes or specific to certain classes and subclasses of immunoglobulins. The main advantage of this method is in the versatility of the conjugate. This reaction is also methodologically simple.
The main stages of an indirect ELISA for the determination of specific antigens (or antibodies) in the sample are as follows:                1. The antigen, or the ligand (antibody) is adsorbed onto a solid phase, and then washed free of unbound components.        2. The free binding sites are blocked. The wash step is repeated.        3. The samples are added to the wells, incubated and then the wells are washed to remove unbound components. Samples serving as positive and negative controls are incubated in parallel wells.        4. The antibody-enzyme or antigen-enzyme conjugate is added at a working dilution, incubated and the unbound components are washed away.        5. The colorimetric substrate is added. The color reaction is stopped by adding a stop solution.        6. The optical density is measure on a reader.        
Under optimum conditions, this method has both a high specificity and a high sensitivity. It can detect nanogram quantities of antigen (or antibody) in serum (or plasma). However, existing methods of immunoassay detection of antigen have a limitation associated with the fact that some antigen in the sample can be present in a complex with other proteins. This complex does not bind to the solid phase, that masks the true concentration of the antigen in the sample. At sufficiently high concentrations of the complex, false negatives may result. To determine the true concentration of antigens, the dissociation of this complex is required. In a previous study of the binding properties of the antigen-antibody complex, it was demonstrated that the use of different organic solvents can increase the sensitivity of the reaction (Mohd. Rehan, HinaYounus, Int. J. of Biol. Macromolecules, Effect of organic solvents on the conformation ant interaction of catalase and anticatalase antibodies).
We have developed a method and a test system with an increased sensitivity of detection of proteolytic products. In the claimed invention, both full-length plasminogen molecules as well as plasminogen fragments of a defined structure are used as antigens and detectors. The claimed invention furthermore uses organic components to detect C-terminal lysine containing proteolysis fragments of immunoglobulin and other protein, which significantly increases the sensitivity of the diagnostic test system.